Arthritis is a joint disorder that involves inflammation of one or more joints. There are over 100 different forms of arthritis. The most common symptom of individuals affected with arthritis is joint pain. Arthritic pain is often constant and may be localized to the affected joint or joints. The pain from arthritis is typically associated with the inflammation that occurs around the joint, the damage to the joint resulting from arthritic disease, the daily wear and tear of the joint, the strain of muscles caused by forceful movements against stiff and painful joints, and fatigue. The direct costs of arthritis in the United States are estimated to be approximately $185.5 billion each year.
Rheumatoid arthritis is a debilitating type of arthritis that affects approximately 1% of the population. Rheumatoid arthritis is a chronic inflammatory disease in animals characterized by the destruction of cartilage and bone, frequently resulting in severe pain. Ultimately, untreated rheumatoid arthritis in patients can result in loss of joint function, reduction in quality of life, and, in extreme cases, morbidity.
The early detection and intervention of arthritis and arthritic disease are important for the successful treatment and preservation of joint mobility and function. For example, biological therapies for arthritis are most effective when they are used to intervene early in the disease process. However, reliable and manageable tools for evaluation of early joint inflammation and the onset and progression of arthritic disease are lacking. Furthermore, although radiographic imaging progression may be able to assess arthritic joint damage, soft tissue inflammation and destruction are not readily detected by radiography. A consensus has not been established for assessment of arthritic events involving inflammatory soft tissue with regard to imaging methods.
Therefore, there exists a need for a method to image a site of arthritis that overcomes the limitations of currently utilized evaluation methods in order to benefit the treatment of arthritis. Accordingly, the present disclosure provides methods of using a nanovesicle comprising a fluorophore which exhibits desirable properties and provides related advantages for the imaging of arthritis and arthritic disease.
The present disclosure demonstrates that a nanovesicle comprising a membrane-associated lysosomal protein (saposin C; “SapC”) incorporated into a phospholipid has a high fusogenic affinity for phosphatidylserine-rich domains on the surfaces of target cell membranes. It is believed that the nanovesicles target surface-exposed phosphatidylserine on the membranes of cells associated with arthritis, allowing for detection of local tissue damage associated with arthritis. In plasma membranes, phosphatidylserine is normally present only on the inner leaflet but is “flipped” to the outer leaflet upon the presence of cell damage. Incorporation of the fluorophore in the nanovesicles allows for the in vivo visualization of the fluorophore in targeted tissue and provides a technique to detect and evaluate the onset and progression of arthritic disease in an animal. Furthermore, the use of the nanovesicle in optical imaging methods provides great promise for analyzing events occurring early in the pathogenesis of arthritis and arthritic disease.
Phosphatidylserine is present not only on cells undergoing apoptosis but also on cells that are injured. In addition, phosphatidylserine has been detected by Annexin V localization to cells that are stressed but not necessarily committed to apoptotic cell death (see Elliott, J. I. et al., “Phosphatidylserine exposure in B lymphocytes: a role for lipid packing,” Blood, 2006; 108:1611-1617; and Balasubramanian, K. et al., “Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis,” J Biol Chem, 2007; 282:18357-18364). Thus, it has been suggested that binding to phosphatidylserine may identify tissues or organs at risk for irreversible injury. In particular, studies of a Collagen-Induced Arthritis (CIA) mouse model using 99mTc-Annexin V, a composition with a high affinity for phosphatidylserine, detected the presence and amount of apoptosis in peripheral joints of arthritic mice using radiographic analysis (see Post, A. M. et al., “Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis,” J Nucl Med, 2002; 43:1359-1365). Thus, the described methods, which utilize a nanovesicle that localizes to cells involved in inflammation and injury in arthritis, provide a valuable tool for the early assessment of arthritic disease and progression of arthritic disease.
The present disclosure describes several advantages compared to current methods utilized for the imaging, detection, and assessment of arthritis in an animal. For instance, embodiments of the present disclosure can be applied via optical imaging, which uses probes that, when excited, emit light that can penetrate the skin more readily than probes with lower emission wavelengths. Thus, the use of optical imaging can facilitate advantageous signal detection in vivo. Some advantages of optical imaging include a decrease in radiation exposure compared to X-ray imaging, a clear targeting and localization of molecular probes associated with disease pathogenesis, and a minimal acquisition time compared to other radiographic methods.
Furthermore, embodiments of the present disclosure can specifically target damaged tissue to enhance imaging of inflammation associated with arthritis. The described nanovesicles comprise a fusogenic protein, saposin C, incorporated into a phospholipid. Saposin C associates with phosphatidylserine on cell membranes by embedding amino and carboxyl-end amphipathic helices into the outer leaflet of membranes (see Qi, X. et al., “Differential membrane interactions of saposins A and C: implications for the functional specificity,” J Biol Chem, 2001; 276:27010-27017).
The following numbered embodiments are contemplated and are non-limiting:
1. A method for imaging a site of arthritis in an animal, the method comprising the step of administering to the animal a nanovesicle comprising saposin C, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a fluorophore,                wherein the site of arthritis is detected by imaging a population of cells at the site of arthritis.        
2. The method of clause 1, wherein the arthritis is an inflammatory arthritis.
3. The method of clause 1, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, traumatic arthritis, bacterial arthritis, post-infectious arthritis, lyme disease (Borreliose), anklyosing spondylilitis, and rubella arthritis.
4. The method of clause 1, wherein the arthritis is rheumatoid arthritis.
5. The method of clause 1, wherein the arthritis is osteoarthritis.
6. The method of any one of clauses 1 to 5, wherein the imaging is optical imaging.
7. The method of any one of clauses 1 to 6, wherein the animal is a mammal.
8. The method of any one of clauses 1 to 7, wherein the animal is a human.
9. The method of any one of clauses 1 to 8, wherein the fluorophore is a near-red fluorophore or an infrared fluorophore.
10. The method of any one of clauses 1 to 8, wherein the fluorophore is selected from the group consisting of CellVue® Maroon (Ex max=647 nm and Em max=667 nm), CellVue® Claret (Ex max=655 nm and Em max=675 nm), CellVue® Plum (Ex max=652 nm and Em max=671 nm), CellVue® Burgundy (Ex max=683 nm and Em max=707 nm), CellVue® Lavendar (Ex max=420 nm and Em max=461 nm), CellVue® NIR815 (Ex max=786 nm and Em max=814 nm), CellVue® NIR780 (Abs max=745 nm and Em max=776 nm), CellVue® Jade (Abs max=478 nm and Em max=508 nm), and CellVue® Red (Ex max=567 nm and Em max=588 nm).
11. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Maroon (CVM).
12. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Claret.
13. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Plum.
14. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Burgundy.
15. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Lavendar.
16. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® NIR815.
17. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® NIR780.
18. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Jade.
19. The method of any one of clauses 1 to 8, wherein the fluorophore is CellVue® Red.
20. The method of any one of clauses 1 to 19, wherein the site of arthritis is selected from the group consisting of hand, foot, knee, elbow, shoulder, hip, and sacroiliac joint.
21. The method of any one of clauses 1 to 11, wherein the site of arthritis is an arthritic joint tissue.
22. The method of any one of clauses 1 to 20, wherein the site of arthritis is a joint.
23. The method of clause 22, wherein the joint is selected from the group consisting of wrist, knee, elbow, forefoot, ankle, subtalar, and synovial joints.
24. The method of any one of clauses 1 to 23, wherein the cells are inflammatory cells.
25. The method of any one of clauses 1 to 24, wherein the cells include an externalized phosphatidylserine on the membrane of the cells.
26. The method of any one of clauses 1 to 25, wherein the cells are associated with arthritis.
27. The method of any one of clauses 1 to 26, wherein the cells are selected from the group consisting of lymphocytes, macrophages, T cells, B cells, natural killer (NK) cells, myeloid cells, fibroblasts, synovial fibroblasts, endothelial cells, mature granulocytes, and neutrophils.
28. The method of any one of clauses 1 to 27, wherein the cells are neutrophils.
29. The method of any one of clauses 1 to 28, wherein the cells express a cell surface marker selected from the group consisting of CD11b, CD19, GR-1, CD31, CD55, and F480.
30. The method of any one of clauses 1 to 29, wherein the cell surface marker is CD11b.
31. The method of any one of clauses 1 to 29, wherein the cell surface marker is CD19.
32. The method of any one of clauses 1 to 29, wherein the cell surface marker is Gr-1.
33. The method of any one of clauses 1 to 29, wherein the cell surface marker is CD31.
34. The method of any one of clauses 1 to 29, wherein the cell surface marker is CD55
35. The method of any one of clauses 1 to 29, wherein the cell surface marker is F480.
36. The method of any one of clauses 1 to 29, wherein the cells are positive for both CD11b and Gr-1.
37. A method for detecting early onset of arthritis in an animal, the method comprising the step of administering to the animal a nanovesicle comprising saposin C, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a fluorophore,                wherein a site of arthritis is detected by imaging a population of cells at the site of arthritis, and        wherein the detection of early onset of arthritis is obtained from the imaging of the population of cells.        
38. The method of clause 37, wherein the arthritis is an inflammatory arthritis.
39. The method of clause 37, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, traumatic arthritis, bacterial arthritis, post-infectious arthritis, lyme disease (Borreliose), anklyosing spondylilitis, and rubella arthritis.
40. The method of clause 37, wherein the arthritis is rheumatoid arthritis.
41. The method of clause 37, wherein the arthritis is osteoarthritis.
42. The method of any one of clauses 37 to 41, wherein the imaging is optical imaging.
43. The method of any one of clauses 37 to 42, wherein the animal is a mammal.
44. The method of any one of clauses 37 to 43, wherein the animal is a human.
45. The method of any one of clauses 37 to 44, wherein the fluorophore is a near-red fluorophore or an infrared fluorophore.
46. The method of any one of clauses 37 to 44, wherein the fluorophore is selected from the group consisting of CellVue® Maroon (Ex max=647 nm and Em max=667 nm), CellVue® Claret (Ex max=655 nm and Em max=675 nm), CellVue® Plum (Ex max=652 nm and Em max=671 nm), CellVue® Burgundy (Ex max=683 nm and Em max=707 nm), CellVue® Lavendar (Ex max=420 nm and Em max=461 nm), CellVue® NIR815 (Ex max=786 nm and Em max=814 nm), CellVue® NIR780 (Abs max=745 nm and Em max=776 nm), CellVue® Jade (Abs max=478 nm and Em max=508 nm), and CellVue® Red (Ex max=567 nm and Em max=588 nm).
47. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Maroon (CVM).
48. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Claret.
49. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Plum.
50. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Burgundy.
51. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Lavendar.
52. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® NIR815.
53. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® NIR780.
54. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Jade.
55. The method of any one of clauses 37 to 44, wherein the fluorophore is CellVue® Red.
56. The method of any one of clauses 37 to 55, wherein the site of arthritis is selected from the group consisting of hand, foot, knee, elbow, shoulder, hip, and sacroiliac joint.
57. The method of any one of clauses 37 to 56, wherein the site of arthritis is an arthritic joint tissue.
58. The method of any one of clauses 37 to 57, wherein the site of arthritis is a joint.
59. The method of clause 58, wherein the joint is selected from the group consisting of wrist, knee, elbow, forefoot, ankle, subtalar, and synovial joints.
60. The method of any one of clauses 37 to 59, wherein the cells are inflammatory cells.
61. The method of any one of clauses 37 to 60, wherein the cells include an externalized phosphatidylserine on the membrane of the cells.
62. The method of any one of clauses 37 to 61, wherein the cells are associated with arthritis.
63. The method of any one of clauses 37 to 62, wherein the cells are selected from the group consisting of lymphocytes, macrophages, T cells, B cells, natural killer (NK) cells, myeloid cells, fibroblasts, synovial fibroblasts, endothelial cells, mature granulocytes, and neutrophils.
64. The method of any one of clauses 37 to 63, wherein the cells are neutrophils.
65. The method of any one of clauses 37 to 64, wherein the cells express a cell surface marker selected from the group consisting of CD11b, CD19, GR-1, CD31, CD55, and F480.
66. The method of any one of clauses 37 to 65, wherein the cell surface marker is CD11b.
67. The method of any one of clauses 37 to 65, wherein the cell surface marker is CD19.
68. The method of any one of clauses 37 to 65, wherein the cell surface marker is Gr-1.
69. The method of any one of clauses 37 to 65, wherein the cell surface marker is CD31.
70. The method of any one of clauses 37 to 65, wherein the cell surface marker is CD55.
71. The method of any one of clauses 37 to 65, wherein the cell surface marker is F480
72. The method of any one of clauses 37 to 65, wherein the cells are positive for both CD11b and Gr-1.
73. A method for assessing disease progression of arthritis in an animal, the method comprising the step of administering to the animal a nanovesicle comprising saposin C, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a fluorophore,                wherein a site of arthritis is detected by imaging a population of cells at the site of arthritis, and        wherein the assessment of disease progression of arthritis is obtained from the imaging of the population of cells.        
74. The method of clause 73, wherein the arthritis is an inflammatory arthritis.
75. The method of clause 73, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, traumatic arthritis, bacterial arthritis, post-infectious arthritis, lyme disease (Borreliose), anklyosing spondylilitis, and rubella arthritis.
76. The method of clause 73, wherein the arthritis is rheumatoid arthritis.
77. The method of clause 73, wherein the arthritis is osteoarthritis.
78. The method of any one of clauses 73 to 77, wherein the imaging is optical imaging.
79. The method of any one of clauses 73 to 78, wherein the animal is a mammal.
80. The method of any one of clauses 73 to 79, wherein the animal is a human.
81. The method of any one of clauses 73 to 80, wherein the fluorophore is a near-red fluorophore or an infrared fluorophore.
82. The method of any one of clauses 73 to 80, wherein the fluorophore is selected from the group consisting of CellVue® Maroon (Ex max=647 nm and Em max=667 nm), CellVue® Claret (Ex max=655 nm and Em max=675 nm), CellVue® Plum (Ex max=652 nm and Em max=671 nm), CellVue® Burgundy (Ex max=683 nm and Em max=707 nm), CellVue® Lavendar (Ex max=420 nm and Em max=461 nm), CellVue® NIR815 (Ex max=786 nm and Em max=814 nm), CellVue® NIR780 (Abs max=745 nm and Em max=776 nm), CellVue® Jade (Abs max=478 nm and Em max=508 nm), and CellVue® Red (Ex max=567 nm and Em max=588 nm).
83. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Maroon (CVM).
84. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Claret.
85. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Plum.
86. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Burgundy.
87. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Lavendar.
88. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® NIR815.
89. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® NIR780.
90. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Jade.
91. The method of any one of clauses 73 to 80, wherein the fluorophore is CellVue® Red.
92. The method of any one of clauses 73 to 91, wherein the site of arthritis is selected from the group consisting of hand, foot, knee, elbow, shoulder, hip, and sacroiliac joint.
93. The method of any one of clauses 73 to 92, wherein the site of arthritis is an arthritic joint tissue.
94. The method of any one of clauses 73 to 93, wherein the site of arthritis is a joint.
95. The method of clause 94, wherein the joint is selected from the group consisting of wrist, knee, elbow, forefoot, ankle, subtalar, and synovial joints.
96. The method of any one of clauses 73 to 95, wherein the cells are inflammatory cells.
97. The method of any one of clauses 73 to 96, wherein the cells include an externalized phosphatidylserine on the membrane of the cells.
98. The method of any one of clauses 73 to 97, wherein the cells are associated with arthritis.
99. The method of any one of clauses 73 to 98, wherein the cells are selected from the group consisting of lymphocytes, macrophages, T cells, B cells, natural killer (NK) cells, myeloid cells, fibroblasts, synovial fibroblasts, endothelial cells, mature granulocytes, and neutrophils.
100. The method of any one of clauses 73 to 99, wherein the cells are neutrophils.
101. The method of any one of clauses 73 to 100, wherein the cells express a cell surface marker selected from the group consisting of CD11b, CD19, GR-1, CD31, CD55, and F480.
102. The method of any one of clauses 73 to 101, wherein the cell surface marker is CD11b.
103. The method of any one of clauses 73 to 101, wherein the cell surface marker is CD19.
104. The method of any one of clauses 73 to 101, wherein the cell surface marker is Gr-1.
105. The method of any one of clauses 73 to 101, wherein the cell surface marker is CD31.
106. The method of any one of clauses 73 to 101, wherein the cell surface marker is CD55.
107. The method of any one of clauses 73 to 101, wherein the cell surface marker is F480
108. The method of any one of clauses 73 to 101, wherein the cells are positive for both CD11b and Gr-1.
109. The method of any one of clauses 73 to 108, wherein the assessment of disease progression of arthritis is used to determine arthritis treatment for the animal.
110. The method of any one of clauses 73 to 109, wherein the assessment of disease progression of arthritis is used to monitor therapeutic response of arthritis treatment to the animal.
111. A method for assessing inflammation of a joint in an animal, the method comprising the step of administering to the animal a nanovesicle comprising saposin C, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a fluorophore,                wherein the inflammation of the joint is detected by imaging a population of cells at the joint, and        wherein the assessment of inflammation is obtained from the imaging of the population of cells.        
112. The method of clause 111, wherein the inflammation of the joint is associated with arthritis.
113. The method of clause 112, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, traumatic arthritis, bacterial arthritis, post-infectious arthritis, lyme disease (Borreliose), anklyosing spondylilitis, and rubella arthritis.
114. The method of clause 112, wherein the arthritis is rheumatoid arthritis.
115. The method of clause 112, wherein the arthritis is osteoarthritis.
116. The method of any one of clauses 111 to 115, wherein the imaging is optical imaging.
117. The method of any one of clauses 111 to 116, wherein the animal is a mammal.
118. The method of any one of clauses 111 to 117, wherein the animal is a human.
119. The method of any one of clauses 111 to 118, wherein the fluorophore is a near-red fluorophore or an infrared fluorophore.
120. The method of any one of clauses 111 to 118, wherein the fluorophore is selected from the group consisting of CellVue® Maroon (Ex max=647 nm and Em max=667 nm), CellVue® Claret (Ex max=655 nm and Em max=675 nm), CellVue® Plum (Ex max=652 nm and Em max=671 nm), CellVue® Burgundy (Ex max=683 nm and Em max=707 nm), CellVue® Lavendar (Ex max=420 nm and Em max=461 nm), CellVue® NIR815 (Ex max=786 nm and Em max=814 nm), CellVue® NIR780 (Abs max=745 nm and Em max=776 nm), CellVue® Jade (Abs max=478 nm and Em max=508 nm), and CellVue® Red (Ex max=567 nm and Em max=588 nm).
121. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Maroon (CVM).
122. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Claret.
123. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Plum.
124. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Burgundy.
125. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Lavendar.
126. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® NIR815.
127. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® NIR780.
128. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Jade.
129. The method of any one of clauses 111 to 118, wherein the fluorophore is CellVue® Red.
130. The method of any one of clauses 111 to 129, wherein the cells include an externalized phosphatidylserine on the membrane of the cells.
131. The method of any one of clauses 111 to 130, wherein the cells are associated with arthritis.
132. The method of any one of clauses 111 to 131, wherein the cells are selected from the group consisting of lymphocytes, macrophages, T cells, B cells, natural killer (NK) cells, myeloid cells, fibroblasts, synovial fibroblasts, endothelial cells, mature granulocytes, and neutrophils.
133. The method of any one of clauses 111 to 132, wherein the cells are neutrophils.
134. The method of any one of clauses 111 to 133, wherein the cells express a cell surface marker selected from the group consisting of CD11b, CD19, GR-1, CD31, CD55, and F480.
135. The method of any one of clauses 111 to 134, wherein the cell surface marker is CD11b.
136. The method of any one of clauses 111 to 134, wherein the cell surface marker is CD19.
137. The method of any one of clauses 111 to 134, wherein the cell surface marker is Gr-1.
138. The method of any one of clauses 111 to 134, wherein the cell surface marker is CD31.
139. The method of any one of clauses 111 to 134, wherein the cell surface marker is CD55
140. The method of any one of clauses 111 to 134, wherein the cell surface marker is F480.
141. The method of any one of clauses 111 to 134, wherein the cells are positive for both CD11b and Gr-1.
142. The method of any one of clauses 111 to 141, wherein the assessment of inflammation is used to determine arthritis treatment for the animal.
143. The method of any one of clauses 111 to 142, wherein the assessment of inflammation is used to monitor therapeutic response of arthritis treatment to the animal.